黄芪根腐病菌腐皮镰刀菌的实时荧光定量PCR检测方法建立  

Fusarium solani Causing Astragalus membranaceus var.mongholicus Root Rot:Establishment of RT-qPCR Detection Method

作　　者：徐英芝 赵丽梅 祖未希[2] 高芬[3] XU Yingzhi;ZHAO Limei;ZU Weixi;GAO Fen(Modern Research Center for Traditional Chinese Medicine,Shanxi University,Taiyuan 030006;Department of Hospitality Management,Taiyuan Tourism College,Taiyuan 030032;Institute of Applied Chemistry,Shanxi University,Taiyuan 030006)

机构地区：[1]山西大学中医药现代研究中心,太原030006 [2]太原旅游职业学院酒店管理系,太原030032 [3]山西大学应用化学研究所,太原030006

出　　处：《中国农学通报》2024年第15期110-116,共7页Chinese Agricultural Science Bulletin

基　　金：山西省基础研究计划资助项目“基于LC-MS非靶向代谢组学和RT-qPCR联用挖掘黄酮类黄芪抗根腐病相关代谢物”(202103021224029);山西省回国留学人员科研资助项目“基于路径分析的山西不同地区仿野生黄芪根腐病发病差异的微生态机制探究”(2022-023)。

摘　　要：本研究成功建立了一种针对黄芪根腐病菌腐皮镰刀菌(Fusarium solani)的实时荧光定量PCR(RT-qPCR)检测方法。该方法基于腐皮镰刀菌翻译延伸因子EF-1α基因序列,设计了特异性引物对FP.F/FP.R,并验证其特异性。建立腐皮镰刀菌的RT-qPCR检测方法;运用所建方法检测黄芪及土壤中腐皮镰刀菌的含量,验证其检测效果。结果表明:设计的引物FP.F/FP.R特异性强,RT-qPCR检测的灵敏度为0.0192 ng/μL,重复性评价显示方法稳定、可靠。应用该RT-qPCR方法,可从黄芪接种发病和疑似发病样本及土壤样本中检测出腐皮镰刀菌。结果表明,3组样本的总阳性检出率均为100%。同时,植株和土壤中腐皮镰刀菌的含量变化与根腐病的严重度基本一致。因此,本研究建立的RT-qPCR方法能够有效地用于黄芪植株及土壤中腐皮镰刀菌的定量检测,为根腐病的早期诊断、预警和病原菌的动态监测提供了重要的技术支持。The study aimed at establishing a rapid and accurate real-time fluorescence quantitative PCR(RT-qPCR)method for the detection of Fusarium solani,the pathogenic fungus of Astragalus membranaceus var.mongholicus(AMM)root rot.Based on the elongation factor-1α(EF-1α)sequence of F.solani,a pair of specific primers FP.F/FP.R was designed and verified for its specificity.The RT-qPCR detection method was established and then used to detect the DNA content of F.solani in AMM plants and soil samples for verifying its detection effect.The results showed that the designed specific primer FP.F/FP.R was highly specific,and the sensitivity of the established RT-qPCR method was 0.0192 ng/μL.Repeatability evaluation showed that the method was stable and reliable.With the mothed,the pathogen F.solani was detected in F.solaniinoculated and suspected diseased AMM plants as well as soil samples,the positive detection rate being 100%for all samples in the three groups.Meanwhile,the changes of F.solani content in AMM plants and soil samples were consistent with the severity of root rot.The results indicated that the RT-qPCR method could be used in quantitative detection of F.solani in AMM plants and soil to provide technical support for the early diagnosis of and warning against root rot,and the dynamic monitoring of pathogens.

关 键 词：蒙古黄芪 根腐病 腐皮镰刀菌 实时荧光定量PCR 早期诊断 动态监测 

分 类 号：S432.44[农业科学—植物病理学]